Abstract
Thioredoxins (Trxs) are oxidoreductase enzymes, present in all organisms, that catalyze the reduction of disulfide bonds in proteins. By applying a calibrated force to a substrate disulfide, the chemical mechanisms of Trx catalysis can be examined in detail at the single-molecule level. Here we use single-molecule force-clamp spectroscopy to explore the chemical evolution of Trx catalysis by probing the chemistry of eight different Trx enzymes. All Trxs show a characteristic Michaelis-Menten mechanism that is detected when the disulfide bond is stretched at low forces, but at high forces, two different chemical behaviors distinguish bacterial-origin from eukaryotic-origin Trxs. Eukaryotic-origin Trxs reduce disulfide bonds through a single-electron transfer reaction (SET), whereas bacterial-origin Trxs show both nucleophilic substitution (SN2) and SET reactions. A computational analysis of Trx structures identifies the evolution of the binding groove as an important factor controlling the chemistry of Trx catalysis.
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Change history
18 November 2009
In the version of this article initially published, Eric A Gaucher’s middle initial was omitted. The error has been corrected in the HTML and PDF versions of the article.
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Acknowledgements
We thank S. Garcia-Manyes and J. Morrone assistance in writing the manuscript and all the Fernandez laboratory members for helpful discussions. This work was supported by the US National Institutes of Health grants HL66030 and HL61228 to J.M.F., grant GMO55090 to J.B., grant GM43340 to B.J.B., grant I.C.E. (Columbia University) to B.J.B. and J.M.F., grant BIO2006-07332 from the Spanish Ministry of Education and Science and FEDER Funds to J.M.S.-R.
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R.P.-J., J.L. and J.M.F. designed the research; R.P.-J., J.L., P.K., I.S.-R. and A.P.W. carried out the experiments; R.P.-J., J.L., P.K., I.S.-R. and J.M.F. conducted the data analysis; D.R.-L. and J.M.S.-R. provided Trx from E. coli; A.C. provided Trxm from pea; A.H. provided human TRX1; A.M.-V. provided human TRX2; K.B. provided Trx1 from P. falciparum; S.-H.C. and J.B. provided Trx2 from E. coli; E. Gelhaye and J.P.J. provided Trxh1 and Trxh3 from poplar; R.P.-J. and E. Gaucher performed the phylogenetic analysis; J.L. and B.J.B. performed computational analysis and molecular dynamics simulations; R.P.-J., J.L., P.K. and J.M.F. wrote the paper; all authors have actively participated in revising the manuscript.
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Perez-Jimenez, R., Li, J., Kosuri, P. et al. Diversity of chemical mechanisms in thioredoxin catalysis revealed by single-molecule force spectroscopy. Nat Struct Mol Biol 16, 890–896 (2009). https://doi.org/10.1038/nsmb.1627
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DOI: https://doi.org/10.1038/nsmb.1627
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